Novel ATPase inhibitor

ABSTRACT

The invention provides a novel human ATPase inhibitor protein (HATPI) and polynucleotides which identify and encode HATPI. The invention also provides genetically engineered expression vectors and host cells containing the nucleic acid sequences encoding HATPI. The invention also provides pharmaceutical compositions containing HATPI or antagonists to HATPI, and in the use of these compositions for the treatment of diseases associated with the expression of HATPI. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding HATPI for the treatment of diseases associated with the expression of HATPI. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, to hybridize to the genomic sequence or transcripts of polynucleotides encoding HATPI or anti-HATPI antibodies which specifically bind to HATPI.

[0001] This application is a continuation application of U.S.application Ser. No. 09/273,135, filed Mar. 19, 1999, which is adivisional of U.S. application Ser. No. 08/893,042, filed Jul. 15, 1997,issued May 25, 1999, as U.S. Pat. No. 5,906,923, entitled ATPASEINHIBITOR, which is a continuation of U.S. application Ser. No.08/725,025, filed Oct. 2, 1996, now abandoned. All of these referencesare hereby expressly incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to nucleic acid and amino acidsequences of a novel human ATPase inhibitor protein and to the use ofthese sequences in the diagnosis, study, prevention, and treatment ofdisease.

BACKGROUND OF THE INVENTION

[0003] The main cellular source of energy for all cell processes is ATP.Most of the cell's ATP is made by H⁺-ATP synthase, a 14 or more subunitprotein complex that spans the mitochondrial inner membrane. One subunitis a small, basic ATPase inhibitor protein, designated as IF. IF hasbeen identified and studied in several species, including ox (Walker J Eet al (1987) Biochemistry 26: 8613-8619) and rat (Higuti T et al (1993)Biochim Biophys Acta 1172: 311-314).

[0004] Bovine IF has a 25 amino acid N-terminal signal sequence that isweakly conserved among several other ATPase complex proteins and maydirect the proteins into the mitochondrial matrix (Walker et al, supra).Amino acid homologies in a highly conserved region in a central portionof IF may constitute an inhibitory motif that is believed to regulatethe activity of ATPase by binding to the C-terminus of the F₁ B-subunit(Polgreen KE et al (1995) Biochim Biophys Acta 1229: 176-180). MammalianIF may exist in two stable conformational forms, a low pH (active) formand a high pH (inactive) form. The interconversion may be due toionizations of histidine residues in the carboxyl half of the proteinresulting in changes in secondary structure (Polgreen et al, supra; SahJ F et al (1993) Biochem Biophys Res Comm 194: 1521-1528).

[0005] There are strong indications for a role of ATPase regulation inischemic injury. In animal experiments, blockage of the coronary arteryfor 10 to 20 minutes was found to result in inhibition of ATPase incardiac mitochondria (Rouslin W (1983) J Biol Chem 258: 9657-9661).Experimental results on cardiac mitochondria suggest that ATPase may bedown-regulated during ischemia by IF binding to ATPase (Rouslin W et al(1990) Arch Biochem Biophys 280: 103-111). Genes for components ofATPase are differentially expressed following ischemia-repurfusion in anon-stress specific manner (Knoll R et al (1996) Biochem Biophys ResCommun 221: 402-407). Thus, changes in mitochondrial ATPase occur duringand after ischemia.

[0006] Heart disease and stroke, the most common causes of death in theUnited States, are ischemic diseases. Ischemic injury can be caused by agradual narrowing of arteries (arteriosclerosis) or complete blockage byblood clots (thrombosis), leading to tissue disfunction or death. Thepolynucleotide sequence and polypeptides of novel ATPase inhibitorswould satisfy a need in the art by providing a new means for theprevention and treatment of ischemic conditions by regulating ATPaseactivity.

SUMMARY

[0007] The invention features a novel human ATPase inhibitor protein(hereinafter referred to as HATPT), characterized as having homology torat ATPase inhibitor (GI 286198) and bovine ATPase inhibitor (GI162713), respectively. Accordingly, the invention features asubstantially purified ATPase inhibitor, having the amino acid sequenceof SEQ ID NO:1, and having characteristics of ATPase inhibitors.

[0008] One aspect of the invention features isolated and substantiallypurified polynucleotides which encode HATPI. In a particular aspect, thepolynucleotides are the nucleotide sequences of SEQ ID NO:2. Inaddition, the invention features polynucleotide sequences that hybridizeunder stringent conditions to SEQ ID NO:2.

[0009] The invention further relates to nucleic acid sequences encodingHATPI, oligonucleotides, peptide nucleic acids (PNA), fragments,portions, or antisense molecules thereof, methods for producing HATPI orfragments thereof, and use of the sequences in expression vectors andhost cells comprising polynucleotides which encode HATPI. The inventionalso relates to antibodies which bind specifically to HATPI andpharmaceutical compositions comprising substantially purified HATPI orfragments thereof, or antagonists of HATPI.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of the novel ATPase inhibitorprotein, HATPI. The alignment was produced using MACDNASIS software(Hitachi Software Engineering Co Ltd, San Bruno Calif.).

[0011]FIGS. 2A and 2B show the northern analysis for Incyte Clone 29416.The northern analysis was produced electronically using LIFESEQ database(Incyte Pharmaceuticals, Palo Alto Calif.).

[0012]FIG. 3 shows the amino acid sequence alignments among HATPI (SEQID NO:1), rat ATPase inhibitor (GI 286198; SEQ ID NO:3), and bovineATPase inhibitor (GI 162713; SEQ ID NO:4). The alignment was producedusing the multisequence alignment program of DNASTAR software (DNAStarInc, Madison Wis.).

[0013]FIG. 4 shows the hydrophobicity plot (generated using MACDNASISsoftware) for HATPI (SEQ ID NO:1). The X axis reflects amino acidposition, and the negative Y axis reflects hydrophobicity (FIGS. 4 and5).

[0014]FIG. 5 shows the hydrophobicity plot for rat ATPase inhibitor (SEQID NO:3).

[0015]FIG. 6 shows the isoelectric plot (generated using MACDNASISsoftware) for HATPI (SEQ ID NO:1).

[0016]FIG. 7 shows the isoelectric plot for rat ATPase inhibitor (SEQ IDNO:3).

DETAILED DESCRIPTION OF THE INVENTION

[0017] Definitions

[0018] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin which may besingle- or double-stranded, and represent the sense or antisense strand.Similarly, amino acid sequence as used herein refers to oligopeptide orprotein sequence.

[0019] “Consensus” as used herein may refer to a nucleic acidsequence 1) which has been resequenced to resolve uncalled bases, 2)which has been extended using XL-PCR (Perkin Elmer, Norwalk Conn.) inthe 5′ or the 3′ direction and resequenced, 3) which has been assembledfrom the overlapping sequences of more than one Incyte clone GCGfragment assembly System, (GCG, Madison Wis.), or 4) which has been bothextended and assembled.

[0020] “Peptide nucleic acid” as used herein refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary (template) strand of nucleic acid (Nielsen PE et al(1993) Anticancer Drug Des 8:53-63).

[0021] As used herein, HATPI refers to the amino acid sequences ofsubstantially purified HATPI obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic or recombinant.

[0022] A “variant” of HATPI is defined as an amino acid sequence that isaltered by one or more amino acids. The variant may have “conservative”changes, wherein a substituted amino acid has similar structural orchemical properties, eg, replacement of leucine with isoleucine. Morerarely, a variant may have “nonconservative” changes, eg, replacement ofa glycine with a tryptophan. Similar minor variations may also includeamino acid deletions or insertions, or both. Guidance in determiningwhich and how many amino acid residues may be substituted, inserted ordeleted without abolishing biological or immunological activity may befound using computer programs well known in the art, for example,DNAStar software.

[0023] An “antagonist” of HATPI refers to a molecule that inhibits thebiological activity of HATPI.

[0024] A “deletion” is defined as a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent.

[0025] An “insertion” or “addition” is that change in an amino acid ornucleotide sequence which has resulted in the addition of one or moreamino acid or nucleotide residues, respectively, as compared to thenaturally occurring HATPI.

[0026] A “substitution” results from the replacement of one or moreamino acids or nucleotides by different amino acids or nucleotides,respectively.

[0027] The term “biologically active” refers to an HATPI havingstructural, regulatory or biochemical functions of a naturally occurringHATPI. Likewise, “immunologically active” defines the capability of thenatural, recombinant or synthetic HATPI, or any oligopeptide thereof, toinduce a specific immune response in appropriate animals or cells and tobind with specific antibodies.

[0028] The term “derivative” as used herein refers to the chemicalmodification of a nucleic acid encoding HATPI or the encoded HATPI.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics ofnatural HATPI.

[0029] As used herein, the term “substantially purified” refers tomolecules, either nucleic or amino acid sequences, that are removed fromtheir natural environment, isolated or separated, and are at least 60%free, preferably 75% free, and most preferably 90% free from othercomponents with which they are naturally associated.

[0030] “Stringency” typically occurs in a range from about Tm-5° C. (5°C. below the Tm of the probe) to about 20° C. to 25° C. below Tm. Aswill be understood by those of skill in the art, a stringencyhybridization can be used to identify or detect identical polynucleotidesequences or to identify or detect similar or related polynucleotidesequences.

[0031] The term “hybridization” as used herein shall include “anyprocess by which a strand of nucleic acid joins with a complementarystrand through base pairing” (Coombs J (1994) Dictionary ofBiotechnoloqy, Stockton Press, New York N.Y.). Amplification as carriedout in the polymerase chain reaction technologies is described inDieffenbach C W and G S Dveksler (1995, PCR Primer, a Laboratory Manual,Cold Spring Harbor Press, Plainview N.Y.).

[0032] Preferred Embodiments

[0033] The invention relates to a novel human ATPase inhibitor and tothe use of the nucleic acid and amino acid sequences in the study,diagnosis, prevention, and treatment of disease. cDNAs encoding aportion of HATPI were found in cDNA libraries from a variety of tissues,and most abundantly in heart tissue (FIGS. 2A and 2B).

[0034] The invention also encompasses HATPI variants. A preferred HATPIvariant is one having at least 80% amino acid sequence similarity to theHATPI amino acid sequence (SEQ ID NO:1), a more preferred HATPI variantis one having at least 90% amino acid sequence similarity to SEQ IDNO:1, and a most preferred HATPI variant is one having at least 95%amino acid sequence similarity to SEQ ID NO:1.

[0035] Nucleic acids encoding the human ATPase inhibitor protein HATPIof the invention were first identified in cDNA, Incyte Clone 29416, froma cDNA library made from a fetal spleen, SPLNFET01, through acomputer-generated search for amino acid sequence alignments. Thefollowing Incyte clones (and cDNA libraries from which they werederived) were extended and assembled to create the consensus sequence(SEQ ID NO:2): 29416 (SPLNFET01); 25530 (PANCNOT01); and 941912(ADRENOT03). HATPI, SEQ ID NO:1, is encoded by the nucleic acid sequenceof SEQ ID NO:2.

[0036] The invention is based, in part, on the chemical and structuralhomology among HATPI, rat ATPase inhibitor (GI 286198; Higuti et al,supra), and bovine ATPase inhibitor (GI 162713; Walker et al, supra;FIG. 3). The novel HATPI is 106 amino acids long and shares 75% identitywith rat ATPase inhibitor. All amino acid sequences that have beenimplicated in APTase inhibitory function, are well conserved in HATPI,including the 5′ signal domain, the inhibitory domain, and histidineresidues implicated in regulation by pH. As illustrated by FIGS. 4, 5,6, and 7, HATPI and rat ATPase inhibitor have similar hydrophobicityplots and isoelectric profiles suggesting similar structural andfunctional properties.

[0037] The HATPI Coding Sequences

[0038] The nucleic acid and deduced amino acid sequences of HATPI areshown in FIGS. 1A and 1B. In accordance with the invention, any nucleicacid sequence which encodes the amino acid sequence of HATPI can be usedto generate recombinant molecules which express HATPI. In a specificembodiment described herein, a nucleotide sequence encoding a portion ofHATPI was first isolated as Incyte Clone 29416 from a fetal spleentissue cDNA library (SPLNFET01).

[0039] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude ofHATPI-encoding nucleotide sequences, some bearing minimal homology tothe nucleotide sequences of any known and naturally occurring gene maybe produced. The invention contemplates each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring HATPI, and all suchvariations are to be considered as being specifically disclosed.

[0040] Although nucleotide sequences which encode HATPI and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HATPI under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HATPI or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic expression host in accordance with the frequency with whichparticular codons are utilized by the host. Other reasons forsubstantially altering the nucleotide sequence encoding HATPI and itsderivatives without altering the encoded amino acid sequences includethe production of RNA transcripts having more desirable properties, suchas a greater half-life, than transcripts produced from the naturallyoccurring sequence.

[0041] It is now possible to produce a DNA sequence, or portionsthereof, encoding an HATPI and its derivatives entirely by syntheticchemistry, after which the synthetic gene may be inserted into any ofthe many available DNA vectors and cell systems using reagents that arewell known in the art at the time of the filing of this application.Moreover, synthetic chemistry may be used to introduce mutations into asequence encoding HATPI or any portion thereof.

[0042] Also included within the scope of the invention arepolynucleotide sequences that are capable of hybridizing to thenucleotide sequence of FIGS. 1A and 1B under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Berger and Kimmel (1987, Guide to Molecular Cloning Techniques,Methods in Enzymology, Vol 152, Academic Press, San Diego Calif.)incorporated herein by reference, and may be used at a definedstringency.

[0043] Altered nucleic acid sequences encoding HATPI which may be usedin accordance with the invention include deletions, insertions orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent HATPI. The proteinmay also show deletions, insertions or substitutions of amino acidresidues which produce a silent change and result in a functionallyequivalent HATPI. Deliberate amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of HATPI is retained. For example, negativelycharged amino acids include aspartic acid and glutamic acid; positivelycharged amino acids include lysine and arginine; and amino acids withuncharged polar head groups having similar hydrophilicity values includeleucine, isoleucine, valine; glycine, alanine; asparagine, glutamine;serine, threonine phenylalanine, and tyrosine.

[0044] Included within the scope of the invention are alleles of HATPI.As used herein, an “allele”, or “allelic sequence” is an alternativeform of HATPI. Alleles result from a mutation, ie, a change in thenucleic acid sequence, and generally produce altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven gene may have none, one or many allelic forms. Common mutationalchanges which give rise to alleles are generally ascribed to naturaldeletions, additions or substitutions of amino acids. Each of thesetypes of changes may occur alone, or in combination with the others, oneor more times in a given sequence.

[0045] Methods for DNA sequencing are well known in the art and employsuch enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (USBiochemical Corp, Cleveland Ohio)), Taq polymerase (Perkin Elmer,Norwalk CT), thermostable T7 polymerase (Amersham, Chicago Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE amplification system marketed by Gibco BRL(Gaithersburg Md.). Preferably, the process is automated with machinessuch as the Hamilton MICROLAB 2200 (Hamilton, Reno Nev.), Peltierthermal cycler (PTC200; MJ Research, Watertown Mass.), and the ABI 377DNA sequencers (Perkin Elmer).

[0046] Extending the Polynucleotide Sequence

[0047] The polynucleotide sequence encoding HATPI may be extendedutilizing partial nucleotide sequence and various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. Gobinda et al (1993; PCR Methods Applic 2:318-22) disclose“restriction-site” polymerase chain reaction (PCR) as a direct methodwhich uses universal primers to retrieve unknown sequence adjacent to aknown locus. First, genomic DNA is amplified in the presence of primerto a linker sequence and a primer specific to the known region. Theamplified sequences are subjected to a second round of PCR with the samelinker primer and another specific primer internal to the first one.Products of each round of PCR are transcribed with an appropriate RNApolymerase and sequenced using reverse transcriptase.

[0048] Inverse PCR can be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia T et al (1988)Nucleic Acids Res 16:8186). The primers may be designed using OLIGO 4.06primer analysis software (1992; National Biosciences Inc, PlymouthMinn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0049] Capture PCR (Lagerstrom M et al (1991) PCR Methods Applic1:111-19) is a method for PCR amplification of DNA fragments adjacent toa known sequence in human and yeast artificial chromosome DNA. CapturePCR also requires multiple restriction enzyme digestions and ligationsto place an engineered double-stranded sequence into an unknown portionof the DNA molecule before PCR.

[0050] Another method which may be used to retrieve unknown sequences isthat of Parker J D et al (1991; Nucleic Acids Res 19:3055-60).Additionally, one can use PCR, nested primers and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0051] Preferred libraries for screening for full length cDNAs are onesthat have been size-selected to include larger cDNAs. Also, randomprimed libraries are preferred in that they will contain more sequenceswhich contain the 5′ and upstream regions of genes. A randomly primedlibrary may be particularly useful if an oligo d(T) library does notyield a full-length cDNA. Genomic libraries are useful for extensioninto the 5′ nontranslated regulatory region.

[0052] Capillary electrophoresis may be used to analyze the size orconfirm the nucleotide sequence of sequencing or PCR products. Systemsfor rapid sequencing are available from Perkin Elmer, BeckmanInstruments (Fullerton Calif.), and other companies. Capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled devise camera. Output/light intensity is converted to electricalsignal using appropriate software (eg. GENOTYPER and SEQUENCE NAVIGATORfrom Perkin Elmer) and the entire process from loading of samples tocomputer analysis and electronic data display is computer controlled.Capillary electrophoresis is particularly suited to the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample. The reproducible sequencing of up to 350 bp of M13phage DNA in 30 min has been reported (Ruiz-Martinez M C et al (1993)Anal Chem 65:2851-2858).

[0053] Expression of the Nucleotide Sequence

[0054] In accordance with the invention, polynucleotide sequences whichencode HATPI, fragments of the polypeptide, fusion proteins orfunctional equivalents thereof may be used in recombinant DNA moleculesthat direct the expression of HATPI in appropriate host cells. Due tothe inherent degeneracy of the genetic code, other DNA sequences whichencode substantially the same or a functionally equivalent amino acidsequence, may be used to clone and express HATPI. As will be understoodby those of skill in the art, it may be advantageous to produceHATPI-encoding nucleotide sequences possessing non-naturally occurringcodons. Codons preferred by a particular prokaryotic or eukaryotic host(Murray E et al (1989) Nuc Acids Res 17:477-508) can be selected, forexample, to increase the rate of HATPI expression or to producerecombinant RNA transcripts having desirable properties, such as alonger half-life, than transcripts produced from naturally occurringsequence.

[0055] The nucleotide sequences of the invention can be engineered inorder to alter an HATPI coding sequence for a variety of reasons,including but not limited to, alterations which modify the cloning,processing and/or expression of the gene product. For example, mutationsmay be introduced using techniques which are well known in the art, eg,site-directed mutagenesis to insert new restriction sites, to alterglycosylation patterns, to change codon preference, to produce splicevariants, etc.

[0056] In another embodiment of the invention, a natural, modified orrecombinant polynucleotides encoding HATPI may be ligated to aheterologous sequence to encode a fusion protein. For example, forscreening of peptide libraries for inhibitors of HATPI activity, it maybe useful to encode a chimeric HATPI protein that is recognized by acommercially available antibody. A fusion protein may also be engineeredto contain a cleavage site located between an HATPI sequence and theheterologous protein sequence, so that the HATPI may be cleaved andpurified away from the heterologous moiety.

[0057] In an alternate embodiment of the invention, the coding sequenceof HATPI may be synthesized, whole or in part, using chemical methodswell known in the art (see Caruthers M H et al (1980) Nuc Acids Res SympSer 215-23, Horn T et al(1980) Nuc Acids Res Symp Ser 225-32, etc).Alternatively, the protein itself could be produced using chemicalmethods to synthesize an HATPI amino acid sequence, whole or in part.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge JY et al (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer) in accordance with the instructionsprovided by the manufacturer.

[0058] The newly synthesized peptide can be substantially by preparativehigh performance liquid chromatography (eg, Creighton (1983) Proteins,Structures and Molecular Principles, W H Freeman and Co, New York N.Y.).The composition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (eg, the Edman degradation procedure; Creighton,supra). Additionally, the amino acid sequence of HATPI, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

[0059] Expression Systems

[0060] In order to express a biologically active HATPI, the nucleotidesequence encoding HATPI or its functional equivalent, is inserted intoan appropriate expression vector, ie, a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0061] Methods which are well known to those skilled in the art can beused to construct expression vectors containing an HATPI coding sequenceand appropriate transcriptional or translational controls. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination or genetic recombination. Such techniques aredescribed in Sambrook et al (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview N.Y. and Ausubel F M et al(1989) Current Protocols in Molecular Biology, John Wiley & Sons, NewYork N.Y.

[0062] A variety of expression vector/host systems may be utilized tocontain and express an HATPI coding sequence. These include but are notlimited to microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (eg, baculovirus); plant cell systemstransfected with virus expression vectors (eg, cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with bacterialexpression vectors (eg, Ti or pBR322 plasmid); or animal cell systems.

[0063] The “control elements” or “regulatory sequences” of these systemsvary in their strength and specificities and are those nontranslatedregions of the vector, enhancers, promoters, and 3′ untranslatedregions, which interact with host cellular proteins to carry outtranscription and translation. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene,LaJolla Calif.) or PSPORT1 (Gibco BRL) and ptrp-lac hybrids and the likemay be used. The baculovirus polyhedrin promoter may be used in insectcells. Promoters or enhancers derived from the genomes of plant cells(eg, heat shock, RUBISCO; and storage protein genes) or from plantviruses (eg, viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from the mammalian genes orfrom mammalian viruses are most appropriate. If it is necessary togenerate a cell line that contains multiple copies of HATPI, vectorsbased on SV40 or EBV may be used with an appropriate selectable marker.

[0064] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for HATPI. For example, whenlarge quantities of HATPI are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be desirable. Such vectors include, but are notlimited to, the multifunctional E. coli cloning and expression vectorssuch as PBLUESCRIPT (stratagene), in which the HATPI coding sequence maybe ligated into the vector in frame with sequences for theamino-terminal Met and the subsequent 7 residues of β-galactosidase sothat a hybrid protein is produced; pIN vectors (Van Heeke & Schuster(1989) J Biol Chem 264:5503-5509); and the like. PGEX vectors (Promega,Madison Wis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems are designedto include heparin, thrombin or factor XA protease cleavage sites sothat the cloned polypeptide of interest can be released from the GSTmoiety at will.

[0065] In the yeast, S. cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase and PGH may be used. For reviews, see Ausubel et al (supra) andGrant et al (1987) Methods in Enzymology 153:516-544.

[0066] In cases where plant expression vectors are used, the expressionof a sequence encoding HATPI may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV (Brisson et al (1984) Nature 310:511-514) may be usedalone or in combination with the omega leader sequence from TMV(Takamatsu et al (1987) EMBO J 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO (Coruzzi et al (1984)EMBO J 3:1671-1680; Broglie et al (1984) Science 224:838-843) or heatshock promoters (Winter J and Sinibaldi R M (1991) Results Probl CellDiffer 17:85-105) may be used. These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. For reviews of such techniques, see Hobbs S or Murry L Ein McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill NewYork N.Y., pp 191-196 or Weissbach and Weissbach (1988) Methods forPlant Molecular Biology, Academic Press, New York N.Y., pp 421-463.

[0067] An alternative expression system which could be used to expressHATPI is an insect system. In one such system, Autographa californicanuclear polyhedrosis virus (AcNPV) is used as a vector to expressforeign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.The HATPI coding sequence may be cloned into a nonessential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of HATPI will render thepolyhedrin gene inactive and produce recombinant virus lacking coatprotein coat. The recombinant viruses are then used to infect S.frugiperda cells or Trichoplusia larvae in which HATPI is expressed(Smith et al (1983) J Virol 46:584; Engelhard E K et al (1994) Proc NatAcad Sci 91:3224-7).

[0068] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, an HATPI coding sequence may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a nonessential E1or E3 region of the viral genome will result in a viable virus capableof expressing HATPI in infected host cells (Logan and Shenk (1984) ProcNatl Acad Sci 81:3655-59). In addition, transcription enhancers, such asthe rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

[0069] Specific initiation signals may also be required for efficienttranslation of an HATPI sequence. These signals include the ATGinitiation codon and adjacent sequences. In cases where HATPI, itsinitiation codon and upstream sequences are inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only coding sequence, ora portion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon must be provided. Furthermore, theinitiation codon must be in the correct reading frame to ensuretranslation of the entire insert. Exogenous transcriptional andinitiation codons can be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate to the cell system in use (Scharf D et al (1994)Results Probl Cell Differ 20:125-62; Bittner et al (1987) Methods inEnzymol 153:516-544).

[0070] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.

[0071] Post-translational processing which cleaves a “prepro” form ofthe protein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, etchave specific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the introduced, foreign protein.

[0072] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress HATPI may be transformed using expression vectors which containviral origins of replication or endogenous expression elements and aselectable marker gene. Following the introduction of the vector, cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clumps of stably transformed cells can be proliferated usingtissue culture techniques appropriate to the cell type.

[0073] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler M et al (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy I et al (1980)Cell 22:817-23) genes which can be employed in tk- or aprt- cells,respectively. Also, antimetabolite, antibiotic, or herbicide resistancecan be used as the basis for selection; for example, dhfr, which confersresistance to methotrexate (Wigler M et al (1980) Proc Natl Acad Sci77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin F et al (1981) J Mol Biol 150:1-14);and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman SCand RC Mulligan (1988) Proc Natl Acad Sci 85:8047-51). Recently, the useof visible markers has gained popularity with such markers asanthocyanins, β glucuronidase and its substrate, GUS, and luciferase andits substrate, luciferin, being widely used not only to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system (Rhodes C Aet al (1995) Methods Mol Biol 55:121-131).

[0074] Identification of Transformants Containing the PolynucleotideSequence

[0075] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionshould be confirmed. For example, if the HATPI is inserted within amarker gene sequence, recombinant cells containing HATPI can beidentified by the absence of marker gene function. Alternatively, amarker gene can be placed in tandem with an HATPI sequence under thecontrol of a single promoter. Expression of the marker gene in responseto induction or selection usually indicates expression of the tandemHATPI as well.

[0076] Alternatively, host cells which contain the coding sequence forHATPI and express HATPI may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay orimmunoassay techniques which include membrane, solution, or chip basedtechnologies for the detection and/or quantification of the nucleic acidor protein.

[0077] The presence of the polynucleotide sequence encoding HATPI can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes, portions, or fragments of polynucleotides encoding HATPI.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the HATPI-encoding sequence todetect transformants containing DNA or RNA encoding HATPI. As usedherein “oligonucleotides” or “oligomers” refer to a nucleic acidsequence of at least about 10 nucleotides and as many as about 60nucleotides, preferably about 15 to 30 nucleotides, and more preferablyabout 20-25 nucleotides which can be used as a probe or amplimer.

[0078] A variety of protocols for detecting and measuring the expressionof HATPI, using either polyclonal or monoclonal antibodies specific forthe protein, are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescentactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson HATPI is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton Ret al (1990, Serological Methods, a Laboratory Manual, APS Press, StPaul Minn.) and Maddox D E et al (1983, J Exp Med 158:1211).

[0079] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding HATPIinclude oligolabeling, nick translation, end-labeling, or PCRamplification using a labeled nucleotide. Alternatively, the HATPIsequence, or any portion of it, may be cloned into a vector for theproduction of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6and labeled nucleotides.

[0080] A number of companies such as Pharmacia Biotech (PiscatawayN.J.), Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio)supply commercial kits and protocols for these procedures. Suitablereporter molecules or labels include those radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241. Also, recombinant immunoglobulins may be produced as shown inU.S. Pat. No. 4,816,567, incorporated herein by reference.

[0081] Purification of HATPI

[0082] Host cells transformed with a nucleotide sequence encoding HATPImay be cultured under conditions suitable for the expression andrecovery of the encoded protein from cell culture. The protein producedby a recombinant cell may be secreted or contained intracellularlydepending on the sequence and/or the vector used. As will be understoodby those of skill in the art, expression vectors containingpolynucleotides encoding HATPI can be designed with signal sequenceswhich direct secretion of HATPI through a prokaryotic or eukaryotic cellmembrane. Other recombinant constructions may join HATPI to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins (Kroll D J et al (1993) DNA Cell Biol12:441-53; cf discussion of vectors infra containing fusion proteins).

[0083] HATPI may also be expressed as a recombinant protein with one ormore additional polypeptide domains added to facilitate proteinpurification. Such purification facilitating domains include, but arenot limited to, metal chelating peptides such as histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor XA or enterokinase (Invitrogen, San Diego Calif.) between thepurification domain and HATPI is useful to facilitate purification. Onesuch expression vector provides for expression of a fusion proteincompromising an HATPI and contains nucleic acid encoding 6 histidineresidues followed by thioredoxin and an enterokinase cleavage site. Thehistidine residues facilitate purification on IMIAC (immobilized metalion affinity chromotography), as described in Porath et al (1992,Protein Expression and Purification 3: 263-281), while the enterokinasecleavage site provides a means for purifying HATPI from the fusionprotein.

[0084] In addition to recombinant production, fragments of HATPI may beproduced by direct peptide synthesis using solid-phase techniques (cfStewart et al (1969) Solid-Phase Peptide Synthesis, W H Freeman Co, SanFrancisco; Merrifield J (1963) J Am Chem Soc 85:2149-2154). In vitroprotein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, using anApplied Biosystems 431A peptide synthesizer (Perkin Elmer, Foster CityCalif.) in accordance with the instructions provided by themanufacturer. Various fragments of HATPI may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

[0085] Uses of HATPI and Polynucleotides Encoding HATPI

[0086] The rationale for use of the novel nucleotide and polypeptidesequences disclosed herein is based in part on the chemical andstructural homology among HATPI, rat ATPase inhibitor (GI 286198; Higutiet al, supra), and bovine ATPase inhibitor (GI 162713; Walker et al,supra) and its expression profile (FIGS. 2A and 2B).

[0087] Mitochondrial ATPase responds to ischemia; therefore, modulatingATPase may protect cells from its harmful effects. Accordingly, thenovel ATPase inhibitor HATPI or an HATPI derivative may be used toprevent or treat adverse effects resulting from ischemia. HATPI can beused to screen for or design specific antagonists, which inhibit theactivity of HATPI. In conditions where HATPI protein activity is notdesirable, a subject could be treated with such an antagonist toinactivate ATPase inhibitor processes.

[0088] HATPI Antibodies

[0089] HATPI-specific antibodies are useful for the diagnosis ofconditions and diseases associated with expression of HATPI. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, single chain, Fab fragments, and fragments produced by a Fabexpression library. Neutralizing antibodies, ie, those which inhibitdimer formation, are especially preferred for diagnostics andtherapeutics.

[0090] HATPI for antibody induction does not require biologicalactivity; however, the protein fragment, or oligopeptide must beantigenic. Peptides used to induce specific antibodies may have an aminoacid sequence consisting of at least five amino acids, preferably atleast 10 amino acids. Preferably, they should mimic a portion of theamino acid sequence of the natural protein and may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of HATPI amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule. Procedures well known in the art can be used forthe production of antibodies to HATPI.

[0091] For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc may be immunized by injection with HATPI or anyportion, fragment or oligopeptide which retains immunogenic properties.

[0092] Depending on the host species, various adjuvants may be used toincrease immunological response. Such adjuvants include but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvumare potentially useful human adjuvants.

[0093] Monoclonal antibodies to HATPI may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Koehler and Milstein(1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosboret al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci80:2026-2030) and the EBV-hybridoma technique (Cole et al (1985)Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New YorkN.Y., pp 77-96).

[0094] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological 35 activity can be used (Morrison et al (1984) Proc Natl AcadSci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda etal (1985) Nature 314:452-454). Alternatively, techniques described forthe production of single chain antibodies (U.S. Pat. No. 4,946,778) canbe adapted to produce HATPI-specific single chain antibodies.

[0095] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G andMilstein C (1991; Nature 349:293-299).

[0096] Antibody fragments which contain specific binding sites for HATPImay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse W D et al (1989) Science 256:1275-1281).

[0097] A variety of protocols for competitive binding orimmunoradiometric assays using either polyclonal or monoclonalantibodies with established specificities are well known in the art.Such immunoassays typically involve the formation of complexes betweenHATPI and its specific antibody and the measurement of complexformation. A two-site, monoclonal-based immunoassay utilizing monoclonalantibodies reactive to two noninterfering epitopes on a specific HATPIprotein is preferred, but a competitive binding assay may also beemployed. These assays are described in Maddox DE et al (1983, J Exp Med158:1211).

[0098] Diagnostic Assays Using HATPI Specific Antibodies

[0099] Particular HATPI antibodies are useful for the diagnosis ofconditions or diseases characterized by expression of HATPI or in assaysto monitor patients being treated with HATPI, agonists or inhibitors.Diagnostic assays for HATPI include methods utilizing the antibody and alabel to detect HATPI in human body fluids or extracts of cells ortissues. The polypeptides and antibodies of the invention may be usedwith or without modification. Frequently, the polypeptides andantibodies will be labeled by joining them, either covalently ornoncovalently, with a reporter molecule. A wide variety of reportermolecules are known, several of which were described above.

[0100] A variety of protocols for measuring HATPI, using eitherpolyclonal or monoclonal antibodies specific for the respective proteinare known in the art. Examples include enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting(FACS). A two-site, monoclonal-based immunoassay utilizing monoclonalantibodies reactive to two non-interfering epitopes on HATPI ispreferred, but a competitive binding assay may be employed. These assaysare described, among other places, in Maddox, D E et al (1983, J Exp Med158:1211).

[0101] In order to provide a basis for diagnosis, normal or standardvalues for HATPI expression must be established. This is accomplished bycombining body fluids or cell extracts taken from normal subjects,either animal or human, with antibody to HATPI under conditions suitablefor complex formation which are well known in the art. The amount ofstandard complex formation may be quantified by comparing variousartificial membranes containing known quantities of HATPI with bothcontrol and disease samples from biopsied tissues. Then, standard valuesobtained from normal samples may be compared with values obtained fromsamples from subjects potentially affected by disease. Deviation betweenstandard and subject values establishes the presence of disease state.

[0102] Drug Screening

[0103] HATPI, its catalytic or immunogenic fragments or oligopeptidesthereof, can be used for screening therapeutic compounds in any of avariety of drug screening techniques. The fragment employed in such atest may be free in solution, affixed to a solid support, borne on acell surface, or located intracellularly. The formation of bindingcomplexes, between HATPI and the agent being tested, may be measured.

[0104] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the HATPI is described in detail in “Determination of AminoAcid Sequence Antigenicity” by Geysen H M, WO Application 84/03564,published on Sep. 13, 1984, and incorporated herein by reference. Insummary, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with fragments of HATPIand washed. Bound HATPI is then detected by methods well known in theart. Purified HATPI can also be coated directly onto plates for use inthe aforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

[0105] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of bindingHATPI specifically compete with a test compound for binding HATPI. Inthis manner, the antibodies can be used to detect the presence of anypeptide which shares one or more antigenic determinants with HATPI.

[0106] Diagnostic and Therapeutic Uses of the Polynucleotide

[0107] A polynucleotide encoding HATPI, or any part thereof, may be usedfor diagnostic and/or therapeutic purposes. For diagnostic purposes,polynucleotides encoding HATPI of this invention may be used to detectand quantitate gene expression in biopsied tissues in which expressionof HATPI may be implicated. The diagnostic assay is useful todistinguish between absence, presence, and excess expression of HATPIand to monitor regulation of HATPI levels during therapeuticintervention. Included in the scope of the invention are oligonucleotidesequences, antisense RNA and DNA molecules, and PNAs.

[0108] Another aspect of the subject invention is to provide forhybridization or PCR probes which are capable of detectingpolynucleotide sequences, including genomic sequences, encoding HATPI orclosely related molecules. The specificity of the probe, whether it ismade from a highly specific region, eg, 10 unique nucleotides in the 5′regulatory region, or a less specific region, eg, especially in the 3′region, and the stringency of the hybridization or amplification(maximal, high, intermediate or low) will determine whether the probeidentifies only naturally occurring sequences encoding HATPI, alleles orrelated sequences.

[0109] Probes may also be used for the detection of related sequencesand should preferably contain at least 50% of the nucleotides from anyof these HATPI encoding sequences. The hybridization probes of thesubject invention may be derived from the nucleotide sequence of SEQ IDNO:2, or from genomic sequence including promoter, enhancer elements andintrons of the naturally occurring HATPI. Hybridization probes may belabeled by a variety of reporter groups, including radionuclides such as32P or 35S, or enzymatic labels such as alkaline phosphatase coupled tothe probe via avidin/biotin coupling systems, and the like.

[0110] Other means for producing specific hybridization probes for DNAsencoding HATPI include the cloning of nucleic acid sequences encodingHATPI or HATPI derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art and are commercially availableand may be used to synthesize RNA probes in vitro by means of theaddition of the appropriate RNA polymerase as T7 or SP6 RNA polymeraseand the appropriate radioactively labeled nucleotides.

[0111] Polynucleotide sequences encoding HATPI may be used for thediagnosis of conditions or diseases with which the expression of HATPIis associated. For example, polynucleotide sequences encoding HATPI maybe used in hybridization or PCR assays of fluids or tissues frombiopsies to detect HATPI expression. The form of such qualitative orquantitative methods may include Southern or northern analysis, dot blotor other membrane-based technologies; PCR technologies; dip stick, pIN,chip and ELISA technologies. All of these techniques are well known inthe art and are the basis of many commercially available diagnostickits.

[0112] The nucleotide sequences encoding HATPI disclosed herein providethe basis for assays that detect activation or induction associated withischemia. The nucleotide sequence encoding HATPI may be labeled bymethods known in the art and added to a fluid or tissue sample from apatient under conditions suitable for the formation of hybridizationcomplexes. After an incubation period, the sample is washed with acompatible fluid which optionally contains a dye (or other labelrequiring a developer) if the nucleotide has been labeled with anenzyme. After the compatible fluid is rinsed off, the dye is quantitatedand compared with a standard. If the amount of dye in the biopsied orextracted sample is significantly elevated over that of a comparablecontrol sample, the nucleotide sequence has hybridized with nucleotidesequences in the sample, and the presence of elevated levels ofnucleotide sequences encoding HATPI in the sample indicates the presenceof the associated disease.

[0113] Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regime in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient. Inorder to provide a basis for the diagnosis of disease, a normal orstandard profile for HATPI expression must be established. This isaccomplished by combining body fluids or cell extracts taken from normalsubjects, either animal or human, with HATPI, or a portion thereof,under conditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fornormal subjects with a dilution series of HATPI run in the sameexperiment where a known amount of a substantially purified HATPI isused. Standard values obtained from normal samples may be compared withvalues obtained from samples from patients afflicted withHATPI-associated diseases. Deviation between standard and subject valuesis used to establish the presence of disease.

[0114] Once disease is established, a therapeutic agent is administeredand a treatment profile is generated. Such assays may be repeated on aregular basis to evaluate whether the values in the profile progresstoward or return to the normal or standard pattern. Successive treatmentprofiles may be used to show the efficacy of treatment over a period ofseveral days or several months.

[0115] PCR, as described in U.S. Pat. Nos. 4,683,195 and 4,965,188,provides additional uses for oligonucleotides based upon the HATPIsequence. Such oligomers are generally chemically synthesized, but theymay be generated enzymatically or produced from a recombinant source.

[0116] Oligomers generally comprise two nucleotide sequences, one withsense orientation (5′→3′) and one with antisense (3′←5′), employed underoptimized conditions for identification of a specific gene or condition.

[0117] The same two oligomers, nested sets of oligomers, or even adegenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

[0118] Additionally, methods which may be used to quantitate theexpression of a particular molecule include radiolabeling (Melby P C etal 1993 J Immunol Methods 159:235-44) or biotinylating (Duplaa C et al1993 Anal Biochem 212:229-236) nucleotides, coamplification of a controlnucleic acid, and standard curves onto which the experimental resultsare interpolated. Quantitation of multiple samples may be speeded up byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or calorimetricresponse gives rapid quantitation. A definitive diagnosis of this typemay allow health professionals to begin aggressive treatment and preventfurther worsening of the condition. Similarly, further assays can beused to monitor the progress of a patient during treatment. Furthermore,the nucleotide sequences disclosed herein may be used in molecularbiology techniques that have not yet been developed, provided the newtechniques rely on properties of nucleotide sequences that are currentlyknown such as the triplet genetic code, specific base pair interactions,and the like.

[0119] Based upon its homology to gene encoding ATPase inhibitorproteins polynucleotide sequences encoding HATPI disclosed herein may beuseful in the treatment of ischemia.

[0120] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensepolynucleotides of the gene encoding HATPI. See, for example, thetechniques described in Sambrook et al (supra) and Ausubel et al(supra).

[0121] The polynucleotides comprising full length cDNA sequence and/orits regulatory elements enable researchers to use sequences encodingHATPI as an investigative tool in sense (Youssoufian H and HF Lodish1993 Mol Cell Biol 13:98-104) or antisense (Eguchi et al (1991) Annu RevBiochem 60:631-652) regulation of gene function. Such technology is nowwell known in the art, and sense or antisense oligomers, or largerfragments, can be designed from various locations along the coding orcontrol regions.

[0122] Genes encoding HATPI can be turned off by transfecting a cell ortissue with expression vectors which express high levels of a desiredHATPI-encoding fragment. Such constructs can flood cells withuntranslatable sense or antisense sequences. Even in the absence ofintegration into the DNA, such vectors may continue to transcribe RNAmolecules until all copies are disabled by endogenous nucleases.Transient expression may last for a month or more with a non-replicatingvector (Mettler I, personal communication) and even longer ifappropriate replication elements are part of the vector system.

[0123] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA or PNA, to thecontrol regions of gene encoding HATPI, ie, the promoters, enhancers,and introns. Oligonucleotides derived from the transcription initiationsite, eg, between −10 and +10 regions of the leader sequence, arepreferred. The antisense molecules may also be designed to blocktranslation of mRNA by preventing the transcript from binding toribosomes. Similarly, inhibition can be achieved using “triple helix”base-pairing methodology. Triple helix pairing compromises the abilityof the double helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA were reviewed by Gee JE et al (In: Huber BEand BI Carr (1994) Molecular and Immunologic Approaches, FuturaPublishing Co, Mt Kisco N.Y.).

[0124] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Withinthe scope of the invention are engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of sequences encoding HATPI.

[0125] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0126] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of RNAmolecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding HATPI. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, antisense cDNA constructsthat synthesize antisense RNA constitutively or inducibly can beintroduced into cell lines, cells or tissues.

[0127] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine and wybutosine as well as acetyl-, methyl-, thio- andsimilarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0128] Methods for introducing vectors into cells or tissues includethose methods discussed infra and which are equally suitable for invivo, in vitro and ex vivo therapy. For ex vivo therapy, vectors areintroduced into stem cells taken from the patient and clonallypropagated for autologous transplant back into that same patient ispresented in U.S. Pat. Nos. 5,399,493 and 5,437,994, disclosed herein byreference. Delivery by transfection and by liposome are quite well knownin the art.

[0129] Furthermore, the nucleotide sequences for HATPI disclosed hereinmay be used in molecular biology techniques that have not yet beendeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including but not limited to suchproperties as the triplet genetic code and specific base pairinteractions.

[0130] Detection and Mapping of Related Polynucleotide Sequences

[0131] The nucleic acid sequence for HATPI can also be used to generatehybridization probes for mapping the naturally occurring genomicsequence. The sequence may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Theseinclude in situ hybridization to chromosomal spreads, flow-sortedchromosomal preparations, or artificial chromosome constructions such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price C M (1993; Blood Rev 7:127-34) and Trask B J (1991;Trends Genet 7:149-54).

[0132] The technique of fluorescent in situ hybridization of chromosomespreads has been described, among other places, in Verma et al (1988)Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, NewYork N.Y. Fluorescent in situ hybridization of chromosomal preparationsand other physical chromosome mapping techniques may be correlated withadditional genetic map data. Examples of genetic map data can be foundin the 1994 Genome Issue of Science (265:1981f). Correlation between thelocation of the gene encoding HATPI on a physical chromosomal map and aspecific disease (or predisposition to a specific disease) may helpdelimit the region of DNA associated with that genetic disease. Thenucleotide sequences of the subject invention may be used to detectdifferences in gene sequences between normal, carrier or affectedindividuals.

[0133] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. For example,a sequence tagged site based map of the human genome was recentlypublished by the Whitehead-MIT Center for Genomic Research (Hudson T Jet al (1995) Science 270:1945-1954). Often the placement of a gene onthe chromosome of another mammalian species such as mouse (WhiteheadInstitute/MIT Center for Genome Research, Genetic Map of the Mouse,Database Release 10, Apr. 28, 1995) may reveal associated markers evenif the number or arm of a particular human chromosome is not known. Newsequences can be assigned to chromosomal arms, or parts thereof, byphysical mapping. This provides valuable information to investigatorssearching for disease genes using positional cloning or other genediscovery techniques. Once a disease or syndrome, such as ataxiatelangiectasia (AT), has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to llq22-23 (Gatti et al(1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier or affected individuals.

[0134] Pharmaceutical Compositions

[0135] The invention relates to pharmaceutical compositions which maycomprise nucleotides, proteins, antibodies, agonists, antagonists, orinhibitors, alone or in combination with at least one other agent, suchas stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. Any of these molecules canbe administered to a patient alone, or in combination with other agents,drugs or hormones, in pharmaceutical compositions where it is mixed withexcipient(s) or pharmaceutically acceptable carriers. In one embodimentof the invention, the pharmaceutically acceptable carrier ispharmaceutically inert.

[0136] Administration of Pharmaceutical Compositions

[0137] Administration of pharmaceutical compositions is accomplishedorally or parenterally. Methods of parenteral delivery include topical,intra-arterial (directly to the tumor), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. In addition to the activeingredients, these pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Maack Publishing Co,Easton Pa.).

[0138] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for ingestion by the patient.

[0139] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0140] Dragee cores are provided with suitable coatings such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, ie, dosage.

[0141] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

[0142] Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active compoundsmay be prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

[0143] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0144] Manufacture and Storage

[0145] The pharmaceutical compositions of the invention may bemanufactured in a manner that known in the art, eg, by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

[0146] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

[0147] After pharmaceutical compositions comprising a compound of theinvention formulated in a acceptable carrier have been prepared, theycan be placed in an appropriate container and labeled for treatment ofan indicated condition. For administration of HATPI, such labeling wouldinclude amount, frequency and method of administration.

[0148] Therapeutically Effective Dose

[0149] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0150] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, eg, of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model is also used to achieve a desirable concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

[0151] A therapeutically effective dose refers to that amount of proteinor its antibodies, antagonists, or inhibitors which ameliorate thesymptoms or condition. Therapeutic efficacy and toxicity of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, eg, ED50 (the dosetherapeutically effective in 50% of the population) and LD50 (the doselethal to 50% of the population). The dose ratio of toxic to therapeuticeffects is the therapeutic index, which can be expressed as the ratio,LD50/ED50. Pharmaceutical compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies is used in formulating a range of dosage for human use.The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0152] The exact dosage is chosen by the individual physician in view ofthe patient to be treated. Dosage and administration are adjusted toprovide sufficient levels of the active moiety or to maintain thedesired effect. Additional factors which may be taken into accountinclude the severity of the disease state, eg, tumor size and location;age, weight and gender of the patient; diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. Long acting pharmaceutical compositionsmight be administered every 3 to 4 days, every week, or once every twoweeks depending on half-life and clearance rate of the particularformulation.

[0153] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0154] It is contemplated, for example, that HATPI or an HATPIderivative can be delivered in a suitable formulation to prevent injurydue to ischemia.

[0155] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0156] I CDNA Library Construction

[0157] The human spleen cell cDNA library was custom constructed byStratagene (catalogue # 937205. Stratagene, La Jolla Calif.). Thestarting cell population is mixed, having been obtained from fetalspleens which have a diverse cell population. Furthermore, the fetalspleens have been pooled from different sources. Poly(A+) RNA (MRNA) waspurified from the spleen cells. cDNA was synthesized from the mRNA.Synthetic adaptor oligonucleotides were ligated onto cDNA ends enablingits insertion into the Uni-ZAP vector system (Stratagene), allowing highefficiency unidirectional (sense orientation) lambda libraryconstruction and the convenience of a plasmid system with blue/whitecolor selection to detect clones with cDNA insertions. Alternativeunidirectional vectors are pcDNA1 (Invitrogen, San Diego Calif.) andpSHlox-1 (Novagen, Madison Wis.).

[0158] II Isolation of CDNA Clones

[0159] The phagemid forms of individual cDNA clones were obtained by thein vivo excision process, in which the host bacterial strain wasco-infected with both the library phage and an fl helper phage.Polypeptides or enzymes derived from both the library-containing phageand the helper phage nicked the DNA, initiated new DNA synthesis fromdefined sequences on the target DNA, and created a smaller, singlestranded circular phagemid DNA molecule that included all DNA sequencesof the PBLUESCRIPT phagemid and the cDNA insert. The phagemid DNA wasreleased from the cells and purified, and used to reinfect fresh hostcells (SOLR, Stratagene) where double-stranded phagemid DNA wasproduced. Because the phagemid carries the gene for β-lactamase, thenewly transformed bacteria were selected on medium containingampicillin.

[0160] Phagemid DNA was also purified using the QIAWELL-8 PlasmidPurification System from the QIAGEN DNA purification system (QIAGENInc., Chatsworth Calif.). This product provides a convenient, rapid andreliable high-throughput method for lysing the bacterial cells andisolating highly purified phagemid DNA using QIAGEN anion-exchange resinparticles with EMPORE membrane technology from 3M in a multiwell format.The DNA was eluted from the purification resin and prepared for DNAsequencing and other analytical manipulations.

[0161] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0162] Each cDNA was compared to sequences in GenBank using a searchalgorithm developed by Applied Biosystems and incorporated into theINHERIT 670 sequence analysis system. In this algorithm, PatternSpecification Language (TRW Inc, Los Angeles Calif.) was used todetermine regions of homology. The three parameters that determine howthe sequence comparisons run were window size, window offset, and errortolerance. Using a combination of these three parameters, the DNAdatabase was searched for sequences containing regions of homology tothe query sequence, and the appropriate sequences were scored with aninitial value. Subsequently, these homologous regions were examinedusing dot matrix homology plots to distinguish regions of homology fromchance matches. Smith-Waterman alignments were used to display theresults of the homology search.

[0163] Peptide and protein sequence homologies were ascertained usingthe INHERIT-670 sequence analysis System in a way similar to that usedin DNA sequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches.

[0164] BLAST, which stands for Basic Local Alignment Search Tool(Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S F et al (1990) JMol Biol 215:403-10), was used to search for local sequence alignments.BLAST produces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs. BLAST is useful for matches which do notcontain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

[0165] An HSP consists of two sequence fragments of arbitrary but equallengths whose alignment is locally maximal and for which the alignmentscore meets or exceeds a threshold or cutoff score set by the user. TheBLAST approach is to look for HSPs between a query sequence and adatabase sequence, to evaluate the statistical significance of anymatches found, and to report only those matches which satisfy theuser-selected threshold of significance. The parameter E establishes thestatistically significant threshold for reporting database sequencematches. E is interpreted as the upper bound of the expected frequencyof chance occurrence of an HSP (or set of HSPs) within the context ofthe entire database search. Any database sequence whose match satisfiesE is reported in the program output.

[0166] IV Northern Analysis

[0167] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabelled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al. supra).

[0168] Analogous computer techniques using BLAST (Altschul S F 1993 and1990, supra) are used to search for identical or related molecules innucleotide databases such as GENBANK or the LIFESEQ database (Incyte,Palo Alto Calif.). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0169] The basis of the search is the product score which is defined as:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0170] and it takes into acccount both the degree of similarity betweentwo sequences and the length of the sequence match. For example, with aproduct score of 40, the match will be exact within a 1-2% error; and at70, the match will be exact. Homologous molecules are usually identifiedby selecting those which show product scores between 15 and 40, althoughlower scores may identify related molecules.

[0171] The results of the search are reported as a list of 1) librariesin which the full length sequence, or parts thereof, is represented 2)the abundance of the sequence, and 3) the percent abundance. Abundancedirectly reflects the number of times a particular transcript is presentin a cDNA library, and percent abundance is abundance divided by thetotal number of sequences examined in the library.

[0172] V Extension of HATPI-encoding Polynucleotides to Full Length orto Recover Regulatory Elements

[0173] Full length HATPI-encoding nucleic acid sequences (SEQ ID NO:2)are used to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5′ sequences fromgenomic libraries. One primer is synthesized to initiate extension inthe antisense direction (XLR) and the other is synthesized to extendsequence in the sense direction (XLF). Primers allow the extension ofthe known HATPI-encoding sequence “outward” generating ampliconscontaining new, unknown nucleotide sequence for the region of interest(U.S. patent application Ser. No. 08/487,112, filed Jun. 7, 1995,specifically incorporated by reference). The initial primers aredesigned from the cDNA using OLIGO 4.06 primer analysis software(National Biosciences), or another appropriate program, to be 22-30nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68°-72° C. Anystretch of nucleotides which would result in hairpin structures andprimer-primer dimerizations is avoided.

[0174] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0175] By following the instructions for the XL-PCR kit (Perkin Elmer)and thoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier thermal cycler (PTC200; MJ Research,Watertown Mass.) and the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (and holding)

[0176] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were selected and cut outof the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK (QIAGEN Inc.). After recovery of theDNA, Klenow enzyme was used to trim single-stranded, nucleotideoverhangs creating blunt ends which facilitate religation and cloning.

[0177] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 160 C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook J et al, supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook J et al, supra)containing 2×Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2×Carb mediumplaced in an individual well of an appropriate, commercially-available,sterile 96-well microtiter plate. The following day, 5 μl of eachovernight culture is transferred into a non-sterile 96-well plate andafter dilution 1:10 with water, 5 μl of each sample is transferred intoa PCR array.

[0178] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

[0179] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid and sequenced.

[0180] VI Labeling and Use of Hybridization Probes

[0181] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 mCi of [γ-³²P] adenosine triphosphate(Amersham, Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN,Boston Mass.). The labeled oligonucleotides are substantially purifiedwith SEPHADEX G-25 super fine resin column (Pharmacia). A portioncontaining 10⁷ counts per minute of each of the sense and antisenseoligonucleotides is used in a typical membrane based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DuPontNEN).

[0182] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (NYTRAN Plus, Schleicher &Schuell, Durham N.H.). Hybridization is carried out for 16 hours at 40°C.

[0183] To remove nonspecific signals, blots are sequentially washed atroom temperature under increasingly stringent conditions up to 0.1×saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARfilm (Kodak, Rochester N.Y.) is exposed to the blots, or the blots areexposed in a Phosphoimager cassette (Molecular Dynamics, SunnyvaleCalif.), hybridization patterns are compared visually.

[0184] VII Antisense Molecules

[0185] The HATPI-encoding sequence, or any part thereof, is used toinhibit in vivo or in vitro expression of naturally occurring HATPI.Although use of antisense oligonucleotides, comprising about 20base-pairs, is specifically described, essentially the same procedure isused with larger cDNA fragments. An oligonucleotide based on the codingsequence of HATPI, as shown in FIGS. 1A and 1B, is used to inhibitexpression of naturally occurring HATPI. The complementaryoligonucleotide is designed from the most unique 5′ sequence as shown inFIGS. 1A and 1B, and used either to inhibit transcription by preventingpromoter binding to the upstream nontranslated sequence or translationof an HATPI-encoding transcript by preventing the ribosome from binding.Using an appropriate portion of the leader and 5′ sequence of SEQ IDNO:2, an effective antisense oligonucleotide includes any 15-20nucleotides spanning the region which translates into the signal orearly coding sequence of the polypeptide as shown in FIG. 1A and 1B.

[0186] VIII Expression of HATPI

[0187] Expression of the HATPI is accomplished by subcloning the cDNAsinto appropriate vectors and transfecting the vectors into host cells.In this case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express HATPI in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Metand the subsequent 7 residues of β-galactosidase. Immediately followingthese eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0188] Induction of an isolated, transfected bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst seven residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length HATPI-encoding sequence. The signal sequencedirects the secretion of HATPI into the bacterial growth media which canbe used directly in the following assay for activity.

[0189] IX HATPI Activity

[0190] HATPI's ATPase inhibitory activity can be measured in vitro bymethods described by Sah et al (supra). Submitochondrial particles areprepared from cells by a procedure described by Klein G et al (1982,Biochim Biophys Acta 681:226-232). Sequences encoding HATPI are placedon an expression vector and HATPI is purified from transformed bacterialcells by standard procedures. Submitochondrial particles (which containATPase) are preincubated with and without HATPI in the presence of Mg⁺⁺and ATP in a BIS TRIS buffered solution at pH 6.0. ATPase is quantitatedby a coupled assay procedure in which ATPase is coupled to β-NADHoxidation using pyruvate kinase and lactate dehydrogenase.

[0191] X Production of HATPI Specific Antibodies

[0192] HATPI substantially purified using PAGE electrophoresis(Sambrook, supra) is used to immunize rabbits and to produce antibodiesusing standard protocols. The amino acid sequence translated from HATPIis analyzed using DNASTAR software (DNAStar Inc.) to determine regionsof high immunogenicity and a corresponding oligopolypeptide issynthesized and used to raise antibodies by means known to those ofskill in the art. Analysis to select appropriate epitopes, such as thosenear the C-terminus or in hydrophilic regions (shown in FIG. 4) isdescribed by Ausubel F M et al (supra).

[0193] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems 431A peptide synthesizer usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma) byreaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS;Ausubel F M et al, supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

[0194] XI Purification of Naturally Occurring HATPI Using SpecificAntibodies

[0195] Naturally occurring or recombinant HATPI is substantiallypurified by immunoaffinity chromatography using antibodies specific forHATPI. An immunoaffinity column is constructed by covalently couplingHATPI antibody to an activated chromatographic resin such asCnBr-activated SEPHAROSE (Pharmacia Biotech). After the coupling, theresin is blocked and washed according to the manufacturer'sinstructions.

[0196] Media containing HATPI is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of HATPI (eg, high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/HATPI binding (eg, a buffer of pH 2-3 or a high concentrationof a chaotrope such as urea or thiocyanate ion), and HATPI is collected.

[0197] XII Identification of Molecules Which Interact with HATPI

[0198] HATPI, or biologically active fragments thereof, are labelledwith ¹²⁵I Bolton-Hunter reagent (Bolton, A E and Hunter, W M (1973)Biochem J 133: 529). Candidate molecules previously arrayed in the wellsof a 96 well plate are incubated with the labelled HATPI, washed, andany wells with labelled HATPI complex are assayed. Data obtained usingdifferent concentrations of HATPI are used to calculate values for thenumber, affinity, and association of HATPI with the candidate molecules.

[0199] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 4 106 amino acids amino acid single linear peptide <Unknown> Consensus1 Met Ala Val Thr Ala Leu Ala Ala Arg Thr Trp Leu Gly Val Trp Gly 1 5 1015 Val Arg Thr Met Gln Ala Arg Gly Phe Gly Ser Asp Gln Ser Glu Asn 20 2530 Val Asp Arg Gly Ala Gly Ser Ile Arg Glu Ala Gly Gly Ala Phe Gly 35 4045 Lys Arg Glu Gln Ala Glu Glu Glu Arg Tyr Phe Arg Ala Gln Ser Arg 50 5560 Glu Gln Leu Ala Ala Leu Lys Lys His His Glu Glu Glu Ile Val His 65 7075 80 His Lys Lys Glu Ile Glu Arg Leu Gln Lys Glu Ile Glu Arg His Lys 8590 95 Gln Lys Ile Lys Met Leu Lys His Asp Asp 100 105 516 base pairsnucleic acid single linear <Unknown> Consensus 2 GNCGTCCCTG CCATTAGCGCGTAACGAGAG ACTGCTTGCT GCGGCAGAGA CGCCAGAGGT 60 GCAGCTCCAG CAGCAATGGCAGTGACGGCG TTGGCGGCGC GGACGTGGCT TGGCGTGTGG 120 GGCGTGAGGA CCATGCAAGCCCGAGGCTTC GGCTCGGATC AGTCCGAGAA TGTCGACCGG 180 GGCGCGGGCT CCATCCGGGAAGCCGGTGGG GCCTTCGGAA AGAGAGAGCA GGCTGAAGAG 240 GAACGATATT TCCGAGCACAGAGTAGAGAA CAACTGGCAG CTTTGAAAAA ACACCATGAA 300 GAAGAAATCG TTCATCATAAGAAGGAGATT GAGCGTCTGC AGAAAGAAAT TGAGCGCCAT 360 AAGCAGAAGA TCAAAATGCTAAAACATGAT GATTAAGTGC ACACCGTGTG CCATAGAATG 420 GCACATGTCA TTGCCCACTTCTGTGTAGAC ATGGTTCTGG TTTAACTAAT ATTTGTCTGT 480 GTGCTACTAA CAGATTATAATAAATTGTCA TCAGTG 516 107 amino acids amino acid single linear peptideGenBank 286198 3 Met Ala Gly Ser Ala Leu Ala Val Arg Ala Arg Leu Gly ValTrp Gly 1 5 10 15 Met Arg Val Leu Gln Thr Arg Gly Phe Gly Ser Asp SerSer Glu Ser 20 25 30 Met Asp Ser Gly Ala Gly Ser Ile Arg Glu Ala Gly GlyAla Phe Gly 35 40 45 Lys Arg Glu Lys Ala Glu Glu Asp Arg Tyr Phe Arg GluLys Thr Arg 50 55 60 Glu Gln Leu Ala Ala Leu Lys Lys His His Glu Asp GluIle Asp His 65 70 75 80 His Ser Lys Glu Ile Glu Arg Leu Gln Lys Gln IleGlu Arg His Lys 85 90 95 Lys Lys Ile Lys Tyr Leu Lys Asn Ser Glu His 100105 109 amino acids amino acid single linear peptide GenBank 162713 4Met Ala Ala Thr Ala Leu Ala Ala Arg Thr Arg Gln Ala Val Trp Ser 1 5 1015 Val Trp Ala Met Gln Gly Arg Gly Phe Gly Ser Glu Ser Gly Asp Asn 20 2530 Val Arg Ser Ser Ala Gly Ala Val Arg Asp Ala Gly Gly Ala Phe Gly 35 4045 Lys Arg Glu Gln Ala Glu Glu Glu Arg Tyr Phe Arg Ala Arg Ala Lys 50 5560 Glu Gln Leu Ala Ala Leu Lys Lys His His Glu Asn Glu Ile Ser His 65 7075 80 His Ala Lys Glu Ile Glu Arg Leu Gln Lys Glu Ile Glu Arg His Lys 8590 95 Gln Ser Ile Lys Lys Leu Lys Gln Ser Glu Asp Asp Asp 100 105

1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: a) a polypeptide comprising an amino acid sequence of SEQ ID NO:1, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence of SEQ ID NO:1, c) a biologically active fragment of a polypeptide having an amino acid sequence of SEQ ID NO:1, and d) an immunogenic fragment of a polypeptide having an amino acid sequence of SEQ ID NO:1.
 2. An isolated polypeptide of claim 1, having a sequence of SEQ ID NO:1.
 3. An isolated polynucleotide encoding a polypeptide of claim
 1. 4. An isolated polynucleotide encoding a polypeptide of claim
 2. 5. An isolated polynucleotide of claim 4, having a sequence of SEQ ID NO:2.
 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim
 3. 7. A cell transformed with a recombinant polynucleotide of claim
 6. 8. A transgenic organism comprising a recombinant polynucleotide of claim
 6. 9. A method for producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.
 10. A method of claim 9, wherein the polypeptide has the sequence of SEQ ID NO:1.
 11. An isolated antibody which specifically binds to a polypeptide of claim
 1. 12. An isolated polynucleotide comprising a sequence selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence of SEQ ID NO:2, b) a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence of SEQ ID NO:2, c) a polynucleotide having a sequence complementary to a polynucleotide of a), d) a polynucleotide having a sequence complementary to a polynucleotide of b) and e) an RNA equivalent of a)-d).
 13. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.
 15. A method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides.
 16. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
 17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.
 18. A composition of claim 17, wherein the polypeptide has an amino acid sequence of SEQ ID NO:1.
 19. A method for treating a disease or condition associated with decreased expression of functional HATPI, comprising administering to a patient in need of such treatment the composition of claim
 17. 20. A method for screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.
 21. A composition comprising an agonist compound identified by a method of claim 20 and a pharmaceutically acceptable excipient.
 22. A method for treating a disease or condition associated with decreased expression of functional HATPI, comprising administering to a patient in need of such treatment a composition of claim
 21. 23. A method for screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.
 24. A composition comprising an antagonist compound identified by a method of claim 23 and a pharmaceutically acceptable excipient.
 25. A method for treating a disease or condition associated with overexpression of functional HATPI, comprising administering to a patient in need of such treatment a composition of claim
 24. 26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim
 1. 27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, said method comprising: a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim
 1. 28. A method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence of claim 5, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
 29. A method for assessing toxicity of a test compound, the method comprising: a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
 30. A diagnostic test for a condition or disease associated with the expression of HATPI in a biological sample, the method comprising: a) combining the biological sample with an antibody of claim 11, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex, and b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.
 31. The antibody of claim 11, wherein the antibody is: a) a chimeric antibody, b) a single chain antibody, c) a Fab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. A composition comprising an antibody of claim 11 and an acceptable excipient.
 33. A method of diagnosing a condition or disease associated with the expression of HATPI in a subject, comprising administering to said subject an effective amount of the composition of claim
 32. 34. A composition of claim 32, wherein the antibody is labeled.
 35. A method of diagnosing a condition or disease associated with the expression of HATPI in a subject, comprising administering to said subject an effective amount of the composition of claim
 34. 36. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 11, the method comprising: a) immunizing an animal with a polypeptide having an amino acid sequence of SEQ ID NO:1, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from said animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which binds specifically to a polypeptide having an amino acid sequence of SEQ ID NO:1.
 37. An antibody produced by a method of claim
 36. 38. A composition comprising the antibody of claim 37 and a suitable carrier.
 39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising: a) immunizing an animal with a polypeptide having an amino acid sequence of SEQ ID NO:1, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide having an amino acid sequence of SEQ ID NO:1.
 40. A monoclonal antibody produced by a method of claim
 39. 41. A composition comprising the antibody of claim 40 and a suitable carrier.
 42. The antibody of claim 11, wherein the antibody is produced by screening a Fab expression library.
 43. The antibody of claim 11, wherein the antibody is produced by screening a recombinant immunoglobulin library.
 44. A method of detecting a polypeptide having an amino acid sequence of SEQ ID NO:1 in a sample, the method comprising: a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide having an amino acid sequence of SEQ ID NO:1 in the sample.
 45. A method of purifying a polypeptide having an amino acid sequence of SEQ ID NO:1 from a sample, the method comprising: a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide having an amino acid sequence of SEQ ID NO:1.
 46. A microarray wherein at least one element of the microarray is a polynucleotide of claim
 13. 47. A method of generating an expression profile of a sample which contains polynucleotides, the method comprising: a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.
 48. An array comprising different nucleotide molecules affixed in distinct physical locations on a solid substrate, wherein at least one of said nucleotide molecules comprises a first oligonucleotide or polynucleotide sequence specifically hybridizable with at least 30 contiguous nucleotides of a target polynucleotide, and wherein said target polynucleotide is a polynucleotide of claim
 12. 49. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.
 50. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide.
 51. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to said target plynucleotide.
 52. An array of claim 48, which is a microarray.
 53. An array of claim 48, further comprising said target polynucleotide hybridized to a nucleotide molecule comprising said first oligonucleotide or polynucleotide sequence.
 54. An array of claim 48, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.
 55. An array of claim 48, wherein each distinct physical location on the substrate contains multiple nucleotide molecules, and the multiple nucleotide molecules at any single distinct physical location have the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another distinct physical location on the substrate.
 56. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:1.
 57. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:2. 